Hepatitis B virus (HBV) is an important human pathogen that affects millions of people worldwide. HBV replicates predominantly in hepatocytes in vivo, resulting in either acute or chronic infection. While acute hepatitis is usually mild, in a substantial portion of infected people the virus is not cleared from the liver. These chronic carriers are at high risk for developing chronic hepatitis, liver cirrhosis, and hepatocarcinoma.
Several vaccines are available that reduce the risk of initial infection with HBV, but many individuals continue to become chronically infected with the virus. In addition, there is a large pool of existing chronic carriers--perhaps as many as 300 million worldwide--who are beyond treatment with vaccines. Development of effective methods and compositions for treatment of HBV infections is an important goal of the pharmaceutical industry. There is thus a need in the art for methods to identify new anti-HBV drugs that can be administered during and after exposure to this viral pathogen.
The HBV genome encodes only four major polypeptides: the surface antigen (S gene), the core antigen (C gene), the viral polymerase, and the X protein. The X protein, designated pX, which is expressed throughout the replication cycle of the virus, transcriptionally activates the HBV enhancer and is important for viral replication. HBV-induced malignant transformation may also require pX; in this case, functional pX is derived from a pX gene that has integrated into the genome of a hepatocyte.
Prior to the present invention, however, the mechanism by which pX activates viral transcription was unknown. Unlike most transcription factors, pX does not bind independently to DNA, and appears to act promiscuously via multiple transcription factors. pX has been reported to alter the DNA binding specificity of CREB and ATF2 (Maguire et al., Science 252:842-844, 1991); to activate Ras-GTP complex formation (Benn et al., Proc. Natl. Acad. Sci. USA 91:10350-10354, 1994); to cause the post-translational modification of c-Jun/c-fos (Natoli et al., Mol. Cell. Biol. 14:989-998, 1994); to possess intrinsic serine kinase activity; to act as a serine proteinase inhibitor (Takada et al., Oncogene 9:341-348, 1994); and to function as a ribo/deoxyribo ATPase (De-Medina et al., Virol. 202:401-407, 1994.)
Heretofore, pX has not been recognized as a target for antiviral therapies, and there is no teaching or suggestion in the prior art of methods for identifying agents that interfere with pX activity in vitro or in vivo. The present invention, by contrast, establishes for the first time that pX activates transcription by binding directly to specific classes of transcription factors, those that contain bZIP domains, thereby promoting dimerization of the factors and increasing their binding to DNA. These surprising findings diverge from all existing reports on the nature of pX activity. Thus, prior to the present invention, there would have been no reason for those skilled in the art to devise screening methods for screening to identify compounds that specifically interfere with pX activity and thus inhibit HBV replication.